The present invention relates to the use of analogs of thrombomodulin (TM) that have the ability to enhance the thrombin-mediated activation of protein C, but which have a significantly reduced ability to activate thrombin-activatable fibrinolysis inhibitor (TAFI), resulting in decreased clotting with an increase in fibrinolysis. These analogs are useful in, for example, antithrombotic therapy. Novel proteins, nucleic acid gene sequences, pharmaceuticals and methods of inhibiting thrombotic activity are disclosed.
There are many disease states that would benefit from treatment with a safe and effective anticoagulant/antithrombotic. The nature of these conditions varies. For example, anticoagulant therapy is useful in acute conditions such as during thrombolytic therapy in myocardial infarction or in treatment of disseminated intravascular coagulation (DIC) associated with, for example, septicemia. Anticoagulants are also useful for less acute conditions, such as chronic use in patients that have received heart valve implants or prophylactic use in surgery patients to reduce the risk of deep venous thrombosis (DVT).
Thrombomodulin is a membrane protein that has demonstrated anticoagulant properties. In humans, it is widely distributed on the endothelium of the vasculature and lymphatics. Its physiological importance has been extensively studied. (See, for example, Esmon et al. (1982) J. Biol. Chem. 257:859-864, Salem et al. (1983) J. Biol. Chem. 259:12246-12251).
Thrombomodulin functions as a receptor for thrombin, a central enzyme in the coagulation cascade. When free, thrombin promotes coagulation both directly by converting fibrinogen to fibrin, indirectly through activation of other proteins in the coagulation cascade (Factors V, VIII and XIII, for example), and through platelet activation. When bound to thrombomodulin, however, the thrombin-thrombomodulin complex is involved in activation of protein C to activated protein C, which then downregulates the coagulation cascade by proteolytically inactivating the essential cofactors Factor Va and Factor VIIIa (Esmon et al., Ann. N. Y. Acad. Sci. (1991), Vol. 614, pp. 30-43) resulting in increased anticoagulant activity. The thrombin-thrombomodulin complex also is involved in activation of thrombin-activatable fibrinolysis inhibitor (TAFI), which leads to an inhibition of fibrinolysis. (See FIG. 1).
The gene encoding native thrombomodulin has been isolated and sequenced from several species, both in its genomic form and as a cDNA clone (Suzuki et al., (1987) EMBO Journal 6:1891-1897; Jackman et al., (1986) Proc. Natl. Acad. Sci. USA 83:8834-8838 and (1987) 84:6425-6429, all of which are herein incorporated by reference). Comparisons with known proteins, such as the LDL receptor, have suggested functional domains (Wen, D., et al., (1987) Biochemistry 26:4350-4357). One study has suggested that the fifth and sixth epidermal growth factor (EGF)-like domains have the capacity to bind thrombin (Kurosawa, S., et al., (1988) J. Biol. Chem. 263:5993-5996); another suggests that EGF-like domains 4, 5, and 6 are sufficient to act as a cofactor for thrombin-mediated protein C activating activity. (Zushi, et al., (1989)J. Biol. Chem. 264:10351-10353). More recent studies have examined the structural elements within the EGF-like domains of thrombomodulin which are required for protein C and thrombin activatable fibrinolysis factor (TAFI) activation by the thrombin-thrombomodulin complex (Kokame et al. (1998) J. Biol. Chem. 273:12135-12139; Wang et al. (1998) Intl. Soc. Fibrin. Throm. pg 11; Wang et al. (2000) J. Biol. Chem. 275:22942-22947). Inhibition of thrombin""s direct procoagulant activity (conversion of fibrinogen to fibrin) can be attributed in part to glycosaminoglycan substituents on the thrombomodulin molecule. (Bourin, M. C. et al., (1986) Pro. Natl. Acad. Sci. USA 83:5924-5928). The O-linked glycosylation domain in thrombomodulin contains potential sites for the addition of these types of sulfated sugars. In addition, thrombomodulin accelerates the direct inhibition of thrombin by natural inhibitors in plasma such as protein C inhibitor (Rezaie et al., (1995) J. Biol. Chem. 270:25336-25339).
Soluble analogs of thrombomodulin that retain most, if not all, of the activities of the native protein have been produced. Furthermore, soluble analogs of thrombomodulin which are resistant to oxidation, resistant to proteolysis, or have in other ways been modified so as to possess a longer half-life within the circulation, have been developed (Glaser et al. These modifications have been described in U.S. Pat. No. 5,256,770 (oxidation resistance), U.S Pat. No. 5,863,760 (protease resistance), and 5,466,668 (altered glycosylation sites), all of which are incorporated herein by reference.
There is a need, however, for new and improved thrombomodulin compositions, both soluble and membrane-bound, which possess altered selectivity of the thrombin-thrombomodulin complex for the substrates protein C and TAFI, resulting in an increase in overall anticoagulant activity. Novel thrombomodulin analogs which, upon binding to thrombin, result in an increased activation of protein C and a decreased activation of TAFI as compared with native thrombomodulin, can fill this need.
In accordance with the present invention, new TM analogs, methods, and compositions are provided which can be used to treat thrombotic disease. Prior art anti-thrombotic compositions produced by recombinant techniques have been studied extensively by the present inventors, leading to improved analogs, which are described in U.S. Pat. Nos. 5,256,770, 5,863,760 and U.S. Pat. No. 5,466,668. The present invention is focused on modification of the substrate specificity of the thrombin-thrombomodulin complex produced when the novel TM analogs of the present invention are used to form this complex.
This invention provides novel TM analogs which, when bound to thrombin, result in a thrombin-thrombomodulin complex which demonstrates greater than 100% activation of protein C and less than 50% activation of TAFI as compared with native thrombomodulin (FIG. 4, SEQ ID NO: 2), the analog having amino acid substitutions at several positions within the 6 EGF-like domains of thrombomodulin, where several is defined as at least two.
Preferred embodiments of these thrombomodulin analogs are those wherein the analog has the amino acid sequence of native thrombomodulin (SEQ ID NO:2) modified at positions 340, 341, or 343 of the c-loop of the third EGF-like domain or at position 381, which is located within EGF-like domain 4, or at a combination of these positions, where the analogs are numbered in accordance with native thrombomodulin (SEQ ID NO: 2). Particularly preferred thrombomodulin analogs contain the modification H381G, combined with one of the following modifications: V340A, D341A or E343A.
A further preferred embodiment of the TM analog is a soluble analog.
A particularly preferred embodiment of the TM analog is one in which the analog is also resistant to oxidation and wherein the methionine at position 388 is replaced with leucine, wherein the analog is numbered in accordance with native thrombomodulin (SEQ ID NO: 2).
Further preferred embodiments of these TM analogs contain additional modifications to provide resistance to protease cleavage and show an altered pattern of glycosylation.
Preferred analogs are ones which contain the following modifications: removal of amino acids 1-3, H381G, M388L, R456G, H457Q, S474A, and any one of the following substitutions: V340A, D341A, or E343A.
Particularly preferred analogs are ones which contain the following modifications: removal of amino acids 1-3, H381G, M388L, R456G, H457Q, S474A, termination at P490, and any one of the following substitutions: V340A, D341A, or E343A.
This invention further provides DNA sequences encoding the TM analogs described above, as well as vectors and host cells to allow production of said TM analogs in prokaryotic or eukaryotic organisms.
This invention further provides methods for treating or preventing thrombotic disease, comprising administering an effective amount of said thrombomodulin analogs to a patient in need of anticoagulant therapy.